The transcriptional mechanisms regulating osteoblast differentiation are not yet fully understood despite the identification of Cbfa1 as a gene necessary and sufficient for this process. Specifically, one critical question that has not been solved yet is to understand the reason for the long delay (3 days) between the onset of Cbfa1 expression during development and the appearance of the first osteoblasts. One possible molecular mechanism to account for this delay would be that a transcriptional inhibitor of osteoblast differentiation may be coexpressed with Cbfa1 transiently during the early stage of skeletal development. We have accumulated genetic and molecular evidence that Twist, a BFILH containing transcription factor acts as a transcriptional and specific inhibitor of Cbfa1. To address this hypothesis we propose to use in this application biochemical, molecular biology and genetic approaches. We believe that these studies will improve our understanding of osteoblast differentiation and early skeleton development. The specific aims are: To perform systematic deletion mutation and domain swapping analyses of Twist to define the domain responsible for its antiosteogenic function. To overexpress either wild-type or mutated forms of Twist during development to delay osteoblast differentiation in vivo. To generate an osteoblast-specific deletion of Twist to initiate osteoblast differentiation earlier during development. To perform biochemical assays to elucidate the molecular bases of the Cbfa 1/Twist interaction.